Fig. 1 ∣. MACHETE allows engineering large genomic deletions.

a, A schematic outline of the MACHETE protocol. A dual selection cassette is integrated via CRISPR HDR into the locus of interest. Cells with stable integration of the cassette are enriched via positive selection, ensuring the presence of the suicide cassette in the population. These KI cells are then used to engineer the intended deletions, which is followed by negative selection. Only those cells that excised the locus will survive the negative selection, greatly enriching for cells with deletions. The cassette excision occurs in a sequence-specific manner, thus eliminating cells with off-target integrations of the cassette. b, MACHETE can be iterated. Given that all exogenous elements are removed at the end of the procedure, deletions (Del) can be sequentially engineered either in the same locus or at a different one. c, MACHETE enables the creation of allelic series of deletions of a locus of interest. Using cells with a KI of the dual selection cassette in the locus of interest, MACHETE allows creating allelic series of deletions of different size (ΔB–C, loss of the region spanning genes B and C; ΔB–E, loss of the region spanning genes B and E; etc.). Image in a adapted from ref. 14, Springer Nature Ltd.